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PUBLICATION 1154 



1963 



CHEMICAL METHODS 
FOR ANALYSIS 
OF FRUIT AND 



VEGETABLE PRODUCTS 




PARTMENT OF AGRICULTURE 



Publication 1154 1963 



CHEMICAL METHODS FOR 

ANALYSIS OF FRUIT 

AND VEGETABLE PRODUCTS 



Compiled 

by 

J. A. Ruck 



Contribution No. B7, Research Station, Summer I and, B.C. 



Research Branch 
CANADA DEPARTMENT OF AGRICULTURE 



First published as Contribution 350, 

Chemistry Division, Science Service, 1956 

Revised — November 1963 



ACKNOWLEDGMENT 

The author is indebted to the following at the Research Station, Sum- 
merland, for help in preparing the manuscript and checking the methods 
recommended in this publication: A. W. Moyls, F.E. Atkinson, J. A. Kitson 
and D. R. MacGregor. 



n 



CONTENTS 

Page 

Introduction v 

Chemicals 1 

Apparatus and Equipment 1 

Calculation of Results 1 

Jams, Jellies and Marmalades 2 

Preparing the material 2 

Water-insoluble solids 2 

Total acidity 2 

Sulfur dioxide — Official Method 3 

Soluble solids 6 

pH 6 

Artificial food dyes 7 

Pectin 8 

Total and reducing sugars — Lane and Eynon Method 9 

Ash 14 

Total solids 14 

Fruit Juices 16 

Ascorbic acid — Indophenol Method 16 

— Colorimetric Method 17 

Total and reducing sugars — Lane and Eynon Method 19 

Total acidity 20 

Specific gravity 20 

Soluble solids 21 

P H 21 

Candied Fruit and Peel 22 

Preparing the material 22 

Total and reducing sugars — Lane and Eynon Method 22 

Total acidity 22 

Sodium benzoate 22 

Sulfur dioxide 22 

Artificial food dyes 22 

Soluble solids 22 

Dehydrated Fruits and Vegetables 23 

Dehydrated apples — Preparing the material 23 

— Moisture 23 

— Sulfur dioxide 23 

Dehydrated potatoes, carrots, etc. — Moisture 23 

iii 



Page 

Mincemeat 24 

Sodium benzoate , 24 

Pickles 26 

Sodium benzoate 26 

Sodium chloride 26 

Wines and Cider 28 

Preparing the material 28 

Alcohol — Pycnometer Method 28 

— By Distillation and Hydrometer 29 

Total acidity 29 

Tannin and coloring matter 30 

Total and reducing sugars 31 

— Total sugar — Dry wines and cider 31 

— Sweet wines and cider 32 

Iron — Colorimetric Method 32 

Total volatile acidity 34 

Volatile acidity — Exclusive of S0 2 35 

Extract — Hydrometer Method 36 

— Oven Method 36 

Sauerkraut 37 

Total acidity 37 

P H 37 

Fruit Preserved in Sulfur Dioxide 37 

Sulfur dioxide - Official Method 37 

- Control Method 37 

Miscellaneous Procedures 39 

Calcium - Official Method 39 

Tannin and coloring matter 40 

Enzyme tests for adequacy of blanching in frozen vegetables ... 41 

Crude fat or ether extract 42 

Estimation of calorie content of diet foods 43 

Appendix 44 

Standard solutions — Acids 44 

— Bases 45 

— Oxidizing and reducing solutions 45 

— Indicators 46 



IV 



INTRODUCTION 

In the Fruit and Vegetable Processing Laboratory, Research Station, 
Summerland, the work includes research with new products, analyses of 
commercial fruit products, and the development of procedures for use in 
establishing grades and standards. In addition, special problems in the fruit 
and vegetable processing industry are studied. 

Chemical methods of analysis are constantly changing through revision, 
and adoption of improved techniques. The methods described in this pub- 
lication are those most commonly used for analyzing the products listed 
and are in current use at the Summerland laboratory. These methods should 
be useful for all units and laboratories engaged in fruit and vegetable 
research. 

Since the printing of the first edition of this manual, several new 
methods have been adopted and the procedures are included in this edition. 

After the outline of each procedure is a list of references that consti- 
tutes acknowledgment of information used in this publication. 



Digitized by the Internet Archive 

in 2012 with funding from 

Agriculture and Agri-Food Canada - Agriculture et Agroalimentaire Canada 



http://www.archive.org/details/chemicalmethodsfOOruck 



CHEMICALS 

All chemicals mentioned in the following procedures are of chemically 
pure (C.P.) grade or equivalent quality. Reagents commonly used, such as 
mineral acids and ammonia, are not included in the lists of reagents for 
individual procedures. When they are mentioned in procedures, C.P. concen- 
trated reagents are to be used. 

Concentrations of solid reagent solutions are indicated in percentages 
on a weight/volume basis: a definite weight of solid reagent is dissolved 
in water and made up to a total volume of 100 ml. Concentrations of liquid 
reagents are indicated in percentages on a volume/volume basis: the given 
volume of the concentrated reagent is diluted to a total volume of 100 ml. 
with water unless otherwise stated. Reagents are sometimes designated on 
a ratio basis such as (4 + 1), which means 4 parts of reagent to 1 part 
water. Water mentioned in any procedure is distilled water. 

Technical grade chemicals usually cost appreciably less than C.P. 
chemicals and where possible they should be used. In the following proce- 
dures these are indicated by the abbreviation (tech.) after the reagent. 



APPARATUS AND EQUIPMENT 

Special apparatus and equipment required for the various procedures 
are given in the description of the analytical method involved. Unless 
otherwise stated, temperatures mentioned are in degrees Centigrade. When 
liquid measurements of solutions and reagents are required to be accurate, 
pipette measurements are specified. Otherwise, a graduated cylinder or 
similar measure is satisfactory. 



CALCULATION OF RESULTS 

The results of analysis of food products are usually expressed on the 
basis of fresh weight, but occasionally on a moisture-free basis. The values 
in the following procedures are expressed as percentages by weight or 
percentages by volume of the original products. 

The weights and aliquots suggested in the procedures are usually 
sufficient where an average amount of the constituent is present. In some 
cases a more suitable weight or aliquot of the sample may have to be 
determined by experiment. 



JAMS, JELLIES AND MARMALADES 

PREPARING THE MATERIAL 

Remove 1/3 to 1/2 of the material for analysis from the container and 
blend for 1 to 2 minutes in a Waring blendor. Take the portion vertically 
through the container to avoid removing excess berries or seeds that may 
have floated to the top. Stir the portion well with a spoon before taking 
each sample for weighing. 

WATER-INSOLUBLE SOLIDS 

Principle 

A weighed sample is boiled with water to extract soluble material. The 
insoluble material is collected on a previously dried, weighed filter paper, 
and washed with hot water. The insoluble solids are then dried in an oven 
and weighed. 

Procedure 

Weigh to the nearest 0.01 gm., on a torsion balance, duplicate 25-gm. 
samples of the blended portion. Transfer each duplicate to a 400-ml. beaker 
with hot water and dilute with additional hot water to about 200 ml. Mix 
and boil gently for 15 to 20 minutes. Transfer one of the duplicate samples 
to a 250-ml. volumetric flask, cool and make up to volume at 20°. (The 
filtrate from this is used later for determining acidity. Mark filtrate "A.") 
Filter separately the sample from the volumetric flask and that from the 
beaker through No. 4 Whatman paper (15.0 cm.) that waspreviously washed 
with hot water, oven-dried for 2 hours at 100°, cooled in a desiccator and 
weighed in a covered weighing dish (7-cm. diam.). Wash with 800 ml. of 
hot water, loosening the water-insoluble solids from filter paper with each 
addition. Transfer filter paper to original weighing dish. Dry overnight at 
100 to 105°, cool in desiccator and weigh. 

Calculations 

% water-insoluble solids - wt. of dry insoluble material x 4. 



TOTAL ACIDITY 

Principle 

Total acidity is determined by titrating an aliquot of the water extract 
with standard sodium hydroxide to pH 8.1 using a pH meter. 



Procedure 

Use filtrate A from the previous water-insoluble solids determination. 
Pipette 50 ml. of the filtrate into 250- ml. beaker. Add about 100 ml. water 
and titrate with 0. IN NaOH to pH 8.1, using a pH meter. Record the amount 
of NaOH required and calculate total acidity. Total acid is expressed as 
the percentage of the predominant acid in the fruit. In small fruits such as 
strawberries, raspberries, black currants, gooseberries and citrus fruits, 
this is taken as citric acid; in plums, cherries, peaches and apricots, as 
malic acid; and in grapes, as tartaric acid. 

Calculations 

Assume the titration required 5.7 ml. 0. IN NaOH and the original 
sample weight was 25 gm. The sample was diluted to 250 ml. and 50-ml. 
aliquot used for titration: 

q, i . l 1 x equiv. wt. of acid x normality of NaOH * titer 

10 wt. of sample 

= 1 x 70.0 x O.lQ x 5.7 

10 5 

= 0.140 x 5.7 

Equivalent wt. of acids: 

Citric (monohydrate) = 70.0 gm. Tartaric = 75.0 gm. 

Malic = 67.0 gm. Acetic = 60.0 gm. 



SULFUR DIOXIDE 

(Official Method) 

Principle 

The sample is acidified and the evolved sulfur dioxide is swept with 
carbon dioxide or nitrogen into cold hydrogen peroxide, where the sulfurous 
acid is oxidized to sulfuric acid. The latter is determined by titration with 
standard sodium hydroxide. 

Reagents 

1. Hydrogen peroxide— 3% solution 

Dilute 200 ml. 30% hydrogen peroxide to about 1,400 ml. in a 
2,000-ml. graduate. Mix by pouring solution back and forth from a 
2,000-ml. beaker to the graduate. Take 100-ml. portion of diluted 
solution (100-ml. graduate) and titrate in a 250-ml. beaker on pH meter 
with 0.1N NaOH to pH 4.1. Do not return this portion to main solution. 
Calculate amount of NaOH required to neutralize the main solution; 
add this amount, stir, check the pH. 



Standardization of H 2 2 solution 

Pipette 10 ml. of the solution into a 100-ml. volumetric flask and 
make up to volume at 20°. Pipette 5 ml. of this diluted solution into 
500-ml. flask, add about 300 ml. water and 10 ml. 6N H 2 S0 4 and titrate 
with 0.1N potassium permanganate to first permanent pink color. If an 
exact 3% solution is required, calculate as follows: 

1 ml. 0.1N KMn0 4 = 0.0017 gm. H 2 2 

If the titration required 9.1 ml. 0.105N KMn0 4 then 

% H 2 2 = 0.017 x 9.1 x normality of KMn0 4 x dilution 
= 0.017 x 9.1 x 0.105 x 200 

Adjust solution to 3.0% with water and always store in a refrig- 
erator. 

2. Bromophenol blue— 0.1% solution 

Dissolve 0.1 gm. in 100 ml. water. 

3. Phenolphthalein indicator— 0. 1% 

Dissolve 0.1 gm. in 50 ml. ethyl alcohol and dilute to 100 ml. with 
water. 

4. Sodium carbonate— saturated solution 

Dissolve enough sodium carbonate (tech.) to prepare a saturated 
solution. Add several drops of phenolphthalein. Discard this solution 
when it becomes decolorized by adsorption of acids in the carbon 
dioxide. 

Apparatus 

The apparatus illustrated in Figure 1 is pyrex throughout. All parts 
have either 24/40 standard taper or 18/9 spherical joints. 

Procedure 

Circulate cold water through condenser. Add from a graduated cylinder 
20 ml. 3% hydrogen peroxide to the Erlenmeyer flask and 5 ml. to the trap. 
Assemble and connect to condenser. Weigh 50 gm. of blended portion and 
rinse into 500-ml. flask through gas inlet tube, using 300 ml. water. Replace 
gas inlet tube immediately, making sure all connections are well greased 
and tight. Remove the gas inlet tube, and slowly add 20 ml. cone. HC1. 
Replace the tube and see if bubbles enter the receiving flasks. If not, 
check joints for leaks. Adjust C0 2 (passed through a gas washing bottle 
filled with sodium carbonate solution) or nitrogen (99.9% pure) to give a 
flow of 15 to 20 bubbles per minute through the tube. Connect the 500-watt 
heater and turn to the high position. In about 5 minutes, when solution 
starts to boil, adjust heater to give a slow boil. Dried fruits or vegetables 
require 1 hour's boiling. Thirty minutes is enough for all other products. 
After the solution boils for the required time, wash the hydrogen peroxide 
solution from the trap into the Erlenmeyer flask. Rinse the trap with water. 




Figure 1. -All-glass apparatus for dete~rminiri9 sulfur dioxide,, A, Gas 
inlet tube* B, 500-ml, round-bottom flask* C, Condenser, 400 mm. jacket 
length, D-E, 18/9 spherical joints. F # Special adapter. G, 250- ml* Erlen- 
meyer flask, H, Trap. 



Add 3 drops of bromophenol blue indicator and titrate with 0. 05N NaOH 
solution to pale sky blue end point, using a 5-ml. microburette (for samples 
very high in S0 2 use a 50-ml. burette and 0.1N NaOH). Run a blank titra- 
tion on 20 ml. of hydrogen peroxide and correct the results accordingly. 

Calculations 

1 ml. 0. IN NaOH = 3.2 mg. S0 2 

If 50-gm. sample required 4.1 ml. 0. 05N NaOH: 

32 x 1000 x normality of NaOH * ml. of NaOH 
p.p.m. S0 2 = "■ wt Q f sam ple 

= 32 x 1000 x Q.05 x 4.1 
50 
References 
Association of Official Agricultural Chemists. Official methods of analysis. 

8th ed. pp. 507-509. Washington, D.C. 1955. 
Shipton, J. Estimation of sulfur dioxide in dried foods. Food Preservation 
Quarterly 14 (3): 54-56. 1954. 

SOLUBLE SOLIDS 

Principle 

Soluble solids are determined with a refractometer equipped with a 
percent sugar scale. 

Procedure 

Take representative samples of a well-mixed portion of jam or jelly 
free from seed and fiber, place on refractometer prisms and read directly 
at 20°. If temperature correction is necessary, use the correction factor 
given in Table 1. It is preferable to have the instrument maintained at 
constant temperature by circulating water through the prisms. For very 
accurate results a correction should be made for water-insoluble solids 
as follows: 

% soluble solids = % solids by refractometer x (100-a) 

100 
where a - % water-insoluble solids. 

PH 

Principle 

The effective acidity is determined by taking a direct reading on a 
pH meter. 

Procedure 

Standardize the pH meter with a pH 4.0 buffer solution. 



Place 50 to 75 gm. of well-mixed portion in a 100-ml. beaker and read 
on pH meter. When the first reading is completed, wipe electrodes with 
small piece of cotton soaked in distilled water. Rinse electrodes with 
water from wash bottle, dry with a piece of filter paper and continue with 
the next determination. 

Table 1. - Temperature Corrections for the Standard Model of 
Sugar Refractometer Calibrated for 20 C. 1 



Tempera- 
ture 


Percentage of dry substance 


5 


10 


15 


20 


25 


30 


35 


40 


45 


50 


55 


60 


65 


70 




Subtract from dry-substance percentages 


15° C. 


.29 


.31 


.33 


.34 


.34 


.35 


.36 


.37' 


.37 


.38 


.39 


.39 


.40 


.40 


16 


.24 


.25 


.26 


.27 


.23 


.28 


.29 


.30 


.30 


.30 


.31 


.31 


.32 


.32 


17 


.18 


.19 


.20 


.21 


.21 


.21 


.22 


.22 


.23 


.23 


.23 


.23 


.24 


.24 


18 


.13 


.13 


.14 


.14 


.14 


.14 


.15 


.15 


.15 


.15 


.16 


.16 


.16 


.16 


19 


.06 


.06 


.07 


.07 


.07 


.07 


.08 


.08 


.03 


.03 


.08 


.08 


.08 


.08 











Add to 


dry-s 


ubstance 


percentages 








21 


.07 


.07 


.07 


.07 


.08 


.03 


.03 


.08 


.03 


.08 


.08 


.08 


.08 


.08 


22 


.13 


.14 


.14 


.15 


.15 


.15 


.15 


.15 


.16 


.16 


.16 


.16 


.16 


.16 


23 


.20 


.21 


.22 


.22 


.23 


.23 


.23 


.23 


.24 


.24 


.24 


.24 


.24 


.24 


24 


.27 


.28 


.29 


.30 


.30 


.31 


.31 


.31 


.31 


.31 


.32 


.32 


.32 


.32 


25 


.35 


.36 


.37 


.38 


.38 


.39 


.40 


.40 


.40 


.40 


.40 


.40 


.40 


.40 


26 


.42 


.43 


.44 


.45 


.46 


.47 


.48 


.48 


.48 


.48 


.48 


.48 


.43 


.48 


27 


.50 


.52 


.53 


.54 


.55 


.55 


.56 


.56 


.56 


.56 


.56 


.56 


.56 


.56 


28 


.57 


.60 


.61 


.63 


.63 


.64 


.64 


.64 


.64 


.64 


.64 


.64 


.64 


.64 


29 


.66 


.68 


.69 


.71 


.72 


.73 


.73 


.73 


.73 


.73 


.73 


.73 


.73 


.73 


30 


.74 


.77 


.78 


.79 


.80 


.80 


.81 


.81 


.81 


.31 


.81 


.81 


.81 


.81 



Proceedings of the Ninth Session of the International Commission for Uniform 
Methods of Sugar Analysis, London. 1936. 



ARTIFICIAL FOOD DYES 

(Water-Soluble Coal Tar Dyes) 

Principle 

The dye is absorbed by a piece of white woolen cloth from an acidified 
solution. Individual pieces of the cloth are moistened separately with cone. 
HC1, cone. H 2 S0 4 , cone. NH 4 0H and 10% NaOH. The colors developed are 
compared with those of known dyes. 

This method is not conclusive but does provide a useful and quick 
estimation of the presence of some artificial food dyes, particularly where 
the dye is present in pure form. 



Procedure 

Dilute 20- to 40-gm. sample with 1 to 3 volumes of water, add 3 or 4 
drops of cone. HC1 and a small piece of white woolen cloth (Nun's veiling). 
Heat to boiling and then cool. Rinse the dyed cloth thoroughly in running 
water, squeeze out excess water and cut into four small pieces; place each 
in a separate depression of a white porcelain spot plate. Moisten separate 
pieces with cone. HC1, cone. H 2 S0 4 , 10% NaOH and cone. NH 4 0H. 

The hue of many coloring matters varies markedly upon treatment with 
acids or alkalies. This variation is also influenced by concentration of 
reagents and quantity of dye present. An unknown dye color should be 
compared with that of a known dye at about the same visual density. Table 
2 shows color changes produced on wool dyed with 0.1 to 0. 5% solutions 
of permitted food dyes. 



Table 2. -Color Reactions Produced on Dyed Fibers by Various Reagents 


Coloring 


Hydrochloric 


Sulfuric 


10% NaOH 


Ammonium 


matter 


acid 


acid 


solution 


hydroxide 


Amaranth 


Slightly darker 


Violet to 
brownish 


Dull brownish 
to orange-red 


Little change 


Erythrosine 


Orange-yellow 


Orange-yellow 


No change 


No change 


Ponceau 3R 


Little change 


Little change 


Dull orange 


Little change 


Ponceau SX 


Deeper red 


Deeper red 


Orange-yellow 


Orange-yellow 


Tartrazine 


Slightly darker 


Slightly darker 


Little change 


Little change 


Naphthol 


Almost decolor- 


Very pale, dull 


No change 


No change 


yellow S 


ized 


brown 






Light green 


Pale orange- 


Yellowish 


Decolorized 


Decolorized 


SF yellowish 


yellow 


brown 






Brilliant blue 


Yellow 


Yellow 


No change 


No change 


FCF 











References 

Association of Official Agricultural Chemists. Official methods of analysis, 7th ed. 
p. 658. Washington, D.C. 1950. 



PECTIN 

(As Calcium Pectate) 1 

Principle 

Pectin is precipitated as calcium pectate from an acid solution by the 
addition of calcium chloride. The calcium pectate precipitate is washed with 
water until chloride-free, then dried and weighed. 

Modification Carre and Haynes method by Dr. Mclnney, Food and Drugs Labora- 
tory, Ottawa, 1944. 



8 



Reagents 

1. Acetic acid— normal solution (approximate) 

Dilute 30 ml. C.P. glacial acetic acid to 500 ml. with water. 

2. Calcium chloride— normal solution (approximate) 

Dissolve 27.5 gm. CaCl 2 (anhydrous) in water and dilute to 500 ml. 

3. Silver nitrate— 1% solution 

Dissolve 1 gm. AgN0 3 in water and dilute to 100 ml. 

Procedure 

Weigh 50 gm. of a blended portion into an 800-ml. beaker. Add about 
400 ml. water and boil for 1 hour, replacing water lost by evaporation. 
Transfer contents of beaker to 500-ml. volumetric flask and make up to 
volume at 20°. Shake well and filter through No. 4 Whatman paper into 500-ml. 
Erlenmeyer flask. 

After mixing the sample by rotating the Erlenmeyer flask, pipette 
duplicate 100-ml. aliquots into 800-ml. beakers. Add 300 ml. water and 10 
ml. IN NaOH from a pipette, stirring constantly, and let stand overnight. 

Add 50 ml. of IN acetic acid, with stirring, and allow to stand for 5 
minutes. Add 25 ml. of IN CaCl 2 solution, with stirring. Allow to stand for 

1 hour. Heat to boiling and boil for 1 minute. Filter through 41H Whatman 
paper (15.0 cm.) that was previously washed with hot water, oven-dried for 

2 hours at 100°, cooled in a desiccator and weighed in a covered weighing 
dish. Wash precipitate with almost boiling water until chloride-free (test 
with AgN0 3 ). Transfer filter paper to original weighing dish. Dry overnight 
at 100°, cool in desiccator and weigh. 



Calculations 



wt. of Ca. pectate x 100 

% Ca pectate = : — ? \ 

r wt. oi sample 



References 

Carre', M. H., and D. Haynes. The estimation of pectin as calcium pectate 
and the application of this method to the determination of the soluble 
pectin in apples. Biochem. J. 16: 60-69. 1922. 



TOTAL AND REDUCING SUGARS 

(Lane and Eynon Method) 

Principle 

Invert sugar reduces the copper in Fehling's solution to red, insoluble 
cuprous oxide. The volume of the unknown sugar solution required to com- 
pletely reduce a measured volume of Fehling's solution is determined by 
titration, using methylene blue as indicator. 



Reagents 

1. Fehling's solution 

Prepare by mixing equal volumes of reagents 2 and 3 immediately 
before use. 

2. Copper sulfate solution 

Dissolve 69.28 gm. CuS0 4 .5H 2 in water, dilute to 1,000 ml. and 
if necessary filter through No. 4 Whatman paper. 

3. Alkaline tartrate solution 

Dissolve 346 gm. Rochelle salt (potassium sodium tartrate, 
KNaC 4 H 4 6 .4H 2 0) and 100 gm. NaOH in water and make up to 1,000 ml. 

4. Methylene blue indicator 

Dissolve 1 gm. methylene blue in 100 ml. of water. 

5. Neutral lead acetate— 45% solution 

Dissolve 225 gm. neutral lead acetate, Pb(C 2 H 3 2 ) 2 .3H 2 0, in water 
and dilute to 500 ml. 

6. Potassium oxalate— 22% solution 

Dissolve 110 gm. potassium oxalate (K 2 C 2 4 .H 2 0) in water and 
dilute to 500 ml. An excess of lead acetate in the sugar solution will 
result in an error in the titration. Determine the exact amount of 
potassium oxalate solution necessary to precipitate the Pb ++ from 
2 ml. of the lead acetate solution as follows: Into each of six 50-ml. 
beakers containing 25 ml. water, pipette 2-ml. aliquots of the lead 
acetate solution. To the beakers add 1.6, 1.7, 1.8, 1.9, 2.0 and 2.1 ml. 
potassium oxalate solution, respectively. Filter each through a 41H 
Whatman paper and collect the filtrates in 50-ml. Erlenmeyer flasks. 
To each of the filtrates add a few drops of potassium oxalate solution. 
The correct amount of potassium oxalate required is the smallest 
amount which, when added to 2 ml. of lead acetate solution, gives a 
negative test for lead in the filtrate, i.e., no precipitate forms. 
The equivalent volumes should be marked on the bottles and employed 
when the solutions are used in sugar determinations. 



Preparing the Standard Sugar Solution 

Weigh 9.5000 gm. of pure sucrose. Transfer to a 400-ml. beaker, add 
100 ml. water and 5 ml. cone. HC1. Let stand 3 days at 20-25° to allow 
inversion to take place. Transfer to a 1,000-ml. flask and make up to 
volume at 20°. The 1% solution thus obtained is stable for several months. 

Neutralize the sugar solution for titration as follows: 
Pipette 50 ml. of the standard invert solution into a 200-ml. volumetric 
flask and add about 100 ml. water. Using phenolphthalein as indicator, 
add 20% NaOH until solution turns pink. Acidify with IN HC1, adding it 
dropwise, until one drop causes the pink color to disappear. Make up to 
volume with water. Titrate against 10 ml. Fehling's solution as described 

10 



under "Standard Method of Titration." The equivalent volume of neutralized 
sugar solution is 20.37. The titration should be within + 0.05 ml., i.e., 
20.32 to 20.42 ml. 

Method of Titration 

With solutions of unknown concentration, the incremental or trial 
method is first employed. When the correct dilutions are established, sub- 
sequent titrations are performed by the standard method. 

The Incremental Method of Titration 

Pipette 10 ml. of the mixed Fehling's solution (reagents 2 and 3) 
into a 250-ml. flask. Add from the burette (Figure 2) 15 ml. of the sugar 
solution or a larger volume if it is known to be insufficient to completely 
reduce the quantity of Fehling's solution used. Mix and heat to boiling on a 
hot plate covered with a clean asbestos-filled gauze. (Note: Wrap adhesive 
tape on neck of flask to make it easier to hold when hot.) Boil for 15 
seconds. If the color remains blue, indicating that the Fehling's solution 
is not completely reduced, make further 3- to 5-ml. additions of the sugar 
solution. Boil the solution for a few seconds after each addition until it is 
judged unsafe (i.e., only the faintest perceptible blue color remains) to 
add more sugar solution without risk of passing the end point. Add 2 drops 
of methylene blue solution and complete the titration by adding the sugar 
solution dropwise until the indicator is completely decolorized. Record 
the volume of solution required. The accuracy of the Incremental Method is 
increased by approaching the end point as rapidly as possible and keeping 
as nearly as possible to a total boiling period of 3 minutes. 

Standard Method of Titration 

Pipette 10 ml. of mixed Fehling's solution (reagents 2 and 3) into 
duplicate 250-ml. Erlenmeyer flasks. Fill the 50-ml. burette with the 
solution to be titrated. Run into the flask almost the whole volume required 
to reduce the Fehling's solution, so that not less than 0.5 ml. or more 
than 1.0 ml. is required later to complete the titration. Mix the contents of 
the flask. Heat to boiling and boil moderately for 2 minutes, then add 2 
drops of the methylene blue solution, taking care not to allow it to touch 
the side of the flask. Complete the titration within 1 minute by adding 2 to 
3 drops of sugar solution at 5- to 10-second intervals, until the indicator 
is completely decolorized. At the end point the boiling liquid assumes the 
brick-red color of precipitated cuprous oxide, which it had before the 
indicator was added. 

Procedure 

Place 50 gm. of the blended jam in an 800-ml. beaker and add 400 ml. 
of water. To prevent inversion of sugars during boiling extraction, neutralize 
the solution to pH 7.5-8 with 0.1N NaOH, using a pH meter. 

11 



= L 



| 2| 

Eli 



E_4fl 



L4£ 



UQ 



W A" 



\5 



F6 I.D. 
RUBBER TUBE 



2-1 .J 



^ 



7"C 
8 



f=G* 



PINCH 
CLAMP 



16 ,D - 
RUBBER TUBE 



TIP OPENING 
I MM. 



Figure 2 -Burette for sugar titration* 



12 



Boil gently for 1 hour, with occasional stirring. Add boiling water to 
maintain the original level. Cool, and transfer to a 500-ml. volumetric 
flask. Make up to volume and filter through No. 4 Whatman paper. Pipette a 
100-ml. aliquot into a 500-ml. volumetric flask. Add 2 ml. of neutral lead 
acetate solution and about 200 ml. of water. Let stand for 10 minutes, then 
precipitate the excess Pb with potassium oxalate as follows: add the 
required amount of potassium oxalate solution as previously determined, 
make up to volume, shake well and filter through 41H Whatman paper. Test 
the filtrate^ for unprecipitated Pb ++ with a drop of potassium oxalate. If 
precipitate forms, add 2 drops of potassium oxalate solution. Refilter and 
retest for Pb ++ . 

Reducing sugars.— Pipette 50 ml. of the clarified solution into a 100- 
or 250-ml. volumetric flask. (The dilution depends on the concentration of 
reducing sugars present.) Make up to volume and titrate by the "Standard 
Method." 

Total sugars.— Pipette 50 ml. of the clarified solution into a 250-ml. 
Erlenmeyer flask. Add 5 gm. of citric acid and 50 ml. of water. Boil gently 
for 10 minutes to invert sucrose, then cool. Transfer to a 250-ml. volumetric 
flask and neutralize as under "Preparation of Standard Sugar Solution." 
Make up to volume and titrate by the "Standard Method." 

Calculations 

Reducing sugar 

o, j . factor x 100 x dilution 

7o reducing sugar = -: -„„ 

& 5 titer 1000 

Using factor from Table 3 

If 24.6 ml. solution were required for titration: 

w , . 51.2 x 100 x 250 

% reducing sugar = ., , ttt^t 
8 8 24.6 1000 

= 51.2 x 25 
24.6 

Total sugar 

If 17.8 ml. solution were required for titration: 

or , ¥ i 50.7 x 25 

% total sugar = ,„ r 
8 17.8 

References 

Atkinson, F.E., and C.C. Strachan. Candying of fruit in B.C. with special 
reference to cherries. Fruit Prod. J. 20: 132, 166, 199, 229, 262, 
310. 1941. 

13 



Table 3. - Factors for 10 ml. of Fehling's Solution to be 
used with Lane and Eynon Volumetric Method 1 



Titer in ml. 


Invert sugar 
no sucrose 


Titer in ml. 


Invert sugar 
no sucrose 


15 


50.5 


33 


51.7 


16 


50.6 


34 


51.7 


17 


50.7 


35 


51.8 


18 


50.8 


36 


51.8 


19 


50.8 


37 


51.9 


20 


50.9 


38 


51.9 


21 


51.0 


39 


52.0 


22 


51.0 


40 


52.0 


23 


51.1 


41 


52.1 


24 


51.2 


42 


52.1 


25 


51.2 


43 


52.2 


26 


51.3 


44 


52.2 


27 


51.4 


45 


52.3 


28 


51.4 


46 


52.3 


29 


51.5 


47 


52.4 


30 


51.5 


48 


52.4 


31 


51.6 


49 


52.5 


32 


51.6 


50 


52.5 



1 Association of Official Agricultural Chemists, Official methods of analysis. 8th 
ed. p. 906, Washington, D.C. 1955. 

ASH 

Principle 

The dried sample is ignited at 525° to a white ash. 

Procedure 

Weigh duplicate 5- to 10-gm. blended samples into 100-ml. flat-bottom 
platinum or porcelain dishes. Heat on water bath or oven at 90° until water 
is expelled. Place slowly in muffle furnace at 525° and leave until white 
ash is obtained. Cool in desiccator and weigh. If black specks appear when 
water is added to the ash, the sample must be redried and placed in the 
furnace until a completely white ash is obtained. The time required varies 
with different products and must be determined by experiment. 

TOTAL SOLIDS 
Principle 

A weighed portion of material is dried in a vacuum oven at a temperature 

not exceeding 70°. Drying time is determined by experiment and is considered 

sufficient when weighings made at 2-hour intervals do not differ by more 

than 3 mg. 



14 



Procedure 

Weigh accurately into large (7 cm.) flat-bottom dishes duplicate 20-gm. 
blended samples, or a quantity that will give not more than 3 to 4 gm. of 
dry material. If necessary, to get a thin layer of the material, add a few ml. 
of water and mix thoroughly. Evaporate to dryness on a water bath and dry 
at 70° in a vacuum oven at 26 inches or higher vacuum until consecutive 
weighings made at intervals of 2 hours do not vary more than 3 mg. Over- 
night drying is usually sufficient for most samples. 



Calculations 



% total solids = dr ^ Wt> x 100 

wt. of sample 



15 



FRUIT JUICES 

ASCORBIC ACID 

(Indophenol Method) 

Principle 

Aliquots in oxalic acid solution are titrated with standardized sodium 
2:6-dichlorophenolindophenol dye to a faint pink color that persists for 5 
to 10 seconds. This method is limited to juices of light color because red 
pigments obscure the end point. 

Reagents 

1. Indophenol dye-0.04% 

Weigh 0.2 gm. sodium 2:6-dichlorophenolindophenol. Dissolve 
in about 200 ml. water, if necessary filter through No. 4 Whatman 
paper into 500-ml. volumetric flask and make up to volume at 20°. 
Store in refrigerator. 

2. Oxalic acid-0.4% 

Dissolve 4 gm. oxalic acid in water and dilute to 1,000 ml. 

Standardization of dye,— Dissolve 2 to 3 gm. potassium iodide in about 
5 ml. water in 50-ml. Erlenmeyer flask (triplicate). Add 15 ml. dye with a 
pipette and then 10 ml. IN HC1. Mix and let stand for 2 minutes. Titrate 
with freshly prepared 0.01N sodium thiosulfate from a microburette (20 ml. 
0.1N in 200-ml. volumetric flask at 20°), using 1 to 2 ml. starch, until there 
is no change in color when one drop or less is added. Complete the titration 
in 1 minute. The dye should be standardized every 48 hours and kept not 
more than two weeks. Store the dye solution in a refrigerator. 

Calculations 

If 15 ml. dye required 3.21 ml. sodium thiosulfate: 

, . . 1 x ml. Na 2 S 2 3 x normality of Na 2 S 2 3 * 88 x 1000 

1 mL ^ = 1000 mTd^ 

= 1 x 3.21 x O.Ql x 88 x 1000 
1000 15 

= 0.188 mg. ascorbic acid 

Procedure 

Shake can well, determine vacuum using a gauge. Pipette 10 ml. juice 
(25 ml. for juices low in vitamin C) into 100-ml. volumetric flask, make up 
to volume with 0.4% oxalic acid and filter through No. 4 Whatman filter 
paper (clarified juices need not be filtered). Pipette a 5- or 10-ml. aliquot 
for titration, depending on amount of ascorbic acid present. Add about 15 
ml. oxalic acid (0.4%) and titrate in a 50-ml. Erlenmeyer flask with 0.04% 

16 



dye to a faint pink end point lasting for 5 to 10 seconds. Titration must be 
completed within 1 minute and the total dye required should not exceed 1.5 
ml. A microburette should be used for the dye. 

Calculations 

mg. ascorbic acid per 100 ml. juice = dye equivalent * titer x dilution. 
If 1.05 ml. dye were required for titration: 
mg. ascorbic acid per 100 ml. juice = 0.188 x 1.05 x 200 

= 39.4 

Rapid Method for Tomatoes 

Blend about 500 gm. of tomatoes in a blendor for not more than 5-7 
seconds. Weigh a 50-gm. sample and using 0.4% oxalic acid transfer to a 
250-ml. volumetric flask. Make up to volume with 0.4% oxalic acid and filter 
through No. 4 Whatman paper. Using a 5- or 10-ml. aliquot of filtrate, titrate 
as above with indophenol dye. 

References 

Bessey, O.A., and C. G. King. The distribution of vitamin C in plant and 
animal tissues, and its determination. J. Biol. Chem. 103: 687-698. 
1933. 

Strachan, C. C. Factors influencing ascorbic acid retention in apple juice. 
Canada Dept. Agr. Pub. 732. Tech. Bull. 40. 1942. 



ASCORBIC ACID 

(Colorimetric Method) 

Principle 

Ascorbic acid is extracted from the material in a Waring blendor using 
oxalic acid. The decolorizing effect of the extracted ascorbic acid on 
indophenol dye is measured with a photoelectric colorimeter. 

Apparatus 

1. Photoelectric colorimeter 

With a Klett-Summerson, use filter No. 540. 

2. Matched tubes 

If matched tubes are not available, mark 4 tubes as follows: 
DW — distilled water 

S — standard (also used later for unknown solution) 
No. 1 
No. 2 
Use these tubes in the required sequence throughout the procedure. 

17 



Reagents 

1. Stock ascorbic acid solution— 0.1% 

Dry ascorbic acid crystals over sulfuric acid. Dissolve 0.2 gm. 
in 0.4% oxalic acid and dilute to 200 ml. with 0.4% oxalic acid. 

2. Working standards (W.S.) 

Take 5, 10, 15, 20 and 25 ml. of stock ascorbic acid solution 
and make each up to 500 ml. with 0.4% oxalic acid. Resulting 
solutions numbered 1 to 5 contain 1, 2, 3, 4 and 5 mg. ascorbic acid 
per 100 ml. respectively. Check each solution against standard 
iodine or 0.04% indophenol dye (use 50 ml. W.S. with 0.01N iodine 
and 20 ml. W.S. with dye). 

3. Dye— sodium 2:6-dichlorophenolindophenol 

Dilute 30 ml. 0.04% dye to 1,000 ml. (12.0 mg./liter) (0.0012%). 

Standardization 

To the four matched tubes add reagents as follows: 

DW - 10 ml. disti lied water 
S - 1 ml. W.S. No. 1 + 9 ml. water. Mix. 

No. 1—1 ml. 0.4% oxalic acid 
No. 2 - 1 ml. W.S. No. 1 
Adjust galvanometer to zero with tube DW in instrument. To tube 
No. 1, add 9 ml. dye, invert to mix, insert into instrument in place of DW 
and take galvanometer reading in 15 seconds. Record as L x . 

Adjust galvanometer to zero with tube S in instrument. To tube No. 2 
add 9 ml. dye, invert to mix, insert into instrument in place of tube S and 
take galvanometer reading in 15 seconds. Record as L 2 . 

In succession, record L x and L 2 readings for each working standard, 
rinsing the tubes with distilled water and drying between each determi- 
nation. 

On graph paper, against concentration of ascorbic acid in mg. per 100 
ml. as abscissa, plot readings L x - L 2 for each working standard. Draw 
standard curve. 

Preparing the Sample 

Weigh 350 gm. 0.4% oxalic acid into blendor jar. Add exactly 50 gm. 
of representative portion of solid material. Blend for 3 minutes and filter 
through No. 4 Whatman paper. If material is juice, pipette 50 ml. into 
250-ml. volumetric flask and make up to volume, using 0.4% oxalic acid. 
Filter if necessary and mark as filtrate. 

Procedure 

Obtain reading L x as described under "Standardization." 
To tube S add 1 ml. filtrate + 9 ml. water, mix and adjust galvanometer 
to zero. 

18 



To tube No. 2 add 1 ml. filtrate + 9 ml. dye, invert to mix, insert into 
instrument in place of tube S and take reading in 15 seconds. Record 
as j jj • 

From the standard curve find the concentration of ascorbic acid in 
mg. per 100 ml. filtrate corresponding to L x - L 2 . 

Calculations 

mg. ascorbic acid per 100 gm. = ascorbic acid in mg. per 100 ml. 

filtrate x dilution. 
Note: Unknown must contain 0.01 to 0.05 mg. ascorbic acid per ml. 

References 

Bessey, O.A. A method for the determination of small quantities of ascorbic 
acid and dehydro ascorbic in turbid and colored solutions in the presence 
of other reducing substances. J. Biol. Chem. 126: 771-784. 1938. 

Evelyn, K. A., H. T. Mallory and C. Rosen. The determination of ascorbic 
acid in urine with the photoelectric colorimeter. J. Biol. Chem. 126: 
645-654. 1938. 

Loeffler, H.J., and J. D. Ponting. Ascorbic acid. Rapid determination in 
fresh, frozen or dehydrated fruits and vegetables. Ind. Eng. Chem. 
Anal. ed. 14: 846-849. 1942. 

TOTAL AND REDUCING SUGARS 

(Lane and Eynon Method) 
Reagents— See "Jams, Jellies and Marmalades." 

Procedure 

Weigh 25 gm. filtered (Whatman No. 4) sample and transfer to 400-ml. 
beaker. Add about 100 ml. water, neutralize to pH 7.5-8 with IN NaOHand 
transfer into 250-ml. volumetric flask. Add 100-200 ml. water and 2 ml. 
lead acetate solution. Shake and let stand for 10 minutes. Add the necessary 
amount of potassium oxalate, bring up to volume with water and filter 
through 41H or No. 5 Whatman paper. Test filtrate with small amount of 
potassium oxalate to determine if lead is absent. Mark filtrate "A." 

Total sugar 

Pipette 50 ml. filtrate A into a 250-ml. Erlenmeyer flask. Add 5 gm. 
citric acid and 50 ml. water. Boil gently for 10 minutes to invert the sucrose, 
then cool. Transfer to a 250-ml. volumetric flask or a 250-ml. beaker and 
neutralize, using phenolphthalein as indicator. Add 20% NaOH until solu- 
tion turns pink. Add IN HC1 dropwise until the pink color disappears. If 
red color obscures the end point, adjust on pH meter to 8.1. Make up to 
volume in a 250-ml. flask and titrate by the '* Standard Method" under 
"Jams, Jellies and Marmalades. " 

19 



Calculations 

m m t , factor x dilution x 100 

70 total sugar = — ,^„ 

5 titer 1000 

= factor (from Table 3) x 5.0 
titer 

Reducing sugar 

For most juices, reducing sugars are low and therefore filtrate A can 
be used directly in the "Standard Method" of titration as directed under 
"Jams, Jellies and Marmalades." 

Calculations 

factor x dilution x 100 



% reducing sugar = 



titer 1000 

= factor (from Table 3) x 1.0 
titer 



TOTAL ACIDITY 

Principle 

The total acidity is determined by titrating a diluted sample of juice 
with standard NaOH to pH 8.1 using a pH meter. 

Procedure 

If juice has not been clarified previously, filter through No. 4 Whatman 
paper. Pipette 10 ml. of juice into 250-ml. beaker. Add about 100 ml. water 
and titrate with 0.1N NaOH to pH 8.1, using a pH meter. 

Calculation 

Calculate percent total acidity as the predominant acid present as 
outlined under acidity of "Jams, Jellies and Marmalades. " 



SPECIFIC GRAVITY 

Principle 

Specific gravity is determined on a sample of juice, using a hydrometer 
at the temperature specified. 

Procedure 

Cool about 200 ml. juice in 250-ml. Erlenmeyer flask to exact temper- 
ature specific for the hydrometer. Rinse hydrometer graduate with about 
50 ml. of cooled juice. Fill graduate with juice, insert the hydrometer and 
take reading. Read the hydrometer at the liquid surface level, not at the 
top of meniscus. Make sure hydrometer is floating freely when read. 

20 



SOLUBLE SOLIDS 

Procedure 

Take soluble solids reading at 20°, using a refractometer. For apple 
juice, the percent soluble solids multiplied by 4 should be about equal to 
the last two figures of the specific gravity reading. 

Example: Soluble solids = 12.0% 

Specific gravity should be close to 1.048 

PH 

Principle 

The pH as measured directly with a pH meter indicates the effective 
acidity of the juice. 

Procedure 

Standardize the pH meter with pH 4.0 buffer. 

Pour juice in 50-ml. beaker and determine pH. This sample can be used 
for evaluating flavor, color, aroma and clarity. 



21 



CANDIED FRUIT AND PEEL 



PREPARING THE MATERIAL 

Pass material for analysis through a food chopper and mix thoroughly 
with a spoon. Products with a liquid portion can be blended. Place in an 
airtight container and store until ready for analysis. 



TOTAL AND REDUCING SUGARS 

(Lane and Eynon Method) 

Procedure 

Proceed as under "Total and Reducing Sugars" in "Jams, Jellies and 
Marmalades." 

TOTAL ACIDITY 

Procedure 

Using a 50-gm. sample, proceed as under "Jams, Jellies and Marma- 
lades." Calculate acidity as citric acid. 

SODIUM BENZOATE 

Procedure 

Proceed as under "Mincemeat." 

SULFUR DIOXIDE 

Procedure 

Using a 50-gm. sample, proceed as under "Jams, Jellies and Marma- 
lades." 

ARTIFICIAL FOOD DYES 

Procedure 

Proceed as under "Jams, Jellies and Marmalades." 

SOLUBLE SOLIDS 

Procedure 

If the product has free syrup, use a portion of the syrup and take a 
soluble solids reading with a refractometer. Temperature correction (Table 
1) should be applied if the instrument is not at 20°. 



22 



DEHYDRATED FRUITS AND VEGETABLES 

Dehydrated Apples 

PREPARING THE MATERIAL 

Pass the material for analysis through a food chopper and mix thor- 
oughly, completing operation as quickly as possible to avoid absorption of 
moisture or loss of sulfur dioxide. Replace material in airtight container 
and hold until ready for analysis. 

MOISTURE 
Procedure 

Spread about 10 gm. of prepared sample over the bottom of a weighed 
aluminum dish provided with a tightly fitted cover (7-cm. diam.). Begin 
weighing with the cover off while adding the sample, until slightly over 
10 gm. Close the dish and weigh accurately and as rapidly as possible. 

Dry for 6 hours at 70° and 26-28 inches vacuum. Remove the dish 
cover during this operation. During the drying, admit to oven a slow current 
of air (2 bubbles per second) dried by passing through sulfuric acid. Replace 
the cover, cool dish in desiccator and weigh. 

Calculations 

~ . loss in wt. inrk 

% moisture = ? r x 100 

wt. of sample 

SULFUR DIOXIDE 
Procedure 

Using a 25-gm. sample, proceed as outlined for "Jams, Jellies and 

Marmalades.' ' Boiling time is 1 hour. 



Dehydrated Potatoes, Carrots, Etc. 

MOISTURE 

Preparing the Material 

Comminute material for analysis in Waring blendor for 1 to 2 minutes. 
Avoid overheating it. Pass through No. 60 mesh sieve and place in an 
airtight container. 

Procedure 

Weigh duplicate 2- to 3-gm. samples into previously dried and weighed 
aluminum dishes (7-cm. diam.). Place in vacuum oven and dry under the 
same conditions as outlined for "Dehydrated Apples." 

Calculations 

v . . loss in wt. irvn 

% moisture = ? = — x 100 

wt. of sample 

23 



MINCEMEAT 

SODIUM BENZOATE 
Principle 

In a sodium chloride solution containing an excess of Na + , by addition 
of sodium hydroxide, benzoic acid is converted into water-soluble sodium 
benzoate. When the sodium benzoate solution is acidified with excess 
hydrochloric acid, water-insoluble benzoic acid is formed. The benzoic 
acid is extracted with chloroform. The chloroform is removed by evaporation 
and the residue containing benzoic acid is dissolved in alcohol and then 
titrated with standard sodium hydroxide. 

Preparing the Material 

Prepare dry materials such as candied fruit by passing them through a 
food chopper; pickles, mincemeat, etc., can be blended. Place materials in 
airtight containers and keep for analysis. 

Procedure 

Weigh a 100-gm. sample and transfer to 500-ml. volumetric flask with 
water (use a powder funnel). Add 10 ml. NaOH (10%) and enough sodium 
chloride to saturate the solution (30 gm. NaCl for every 100 ml. solution). 
Adjust volume of liquid to about 400 ml. and allow to stand 2 hours, with 
frequent shaking. 

Make up to volume with water and filter through No. 4 Whatman paper 
into a 600-ml. beaker. Pipette 100 ml. filtered extract into 500-ml. shaking 
bottles and neutralize withHCl (l + 3). If 10 ml. NaOH was added previously, 
the amount of HC1 required is 2 ml. Add 5 ml. excess or a total of 7 ml. 
HC1 (1 + 3). Add 50 ml. chloroform to each bottle, close cap and shake 
gently, releasing the cap periodically to release any pressure. Shake and 
let stand alternately for 30 minutes. Shaking should be sufficient for proper 
mixing but not violent enough to cause an emulsion. This point varies with 
the product. 

After 30 minutes, transfer solution slowly to a 500-ml. separatory 
funnel. When layers have separated, draw off 25 ml. of the lower layer and 
transfer to a 250-ml. beaker. Allow to stand at room temperature until 
chloroform has evaporated. Dissolve residue by adding 50 ml. alcohol 
(4 + 1). Add 50 ml. water and titrate on a pH meter to pH 8.1, using 0.05N 
sodium hydroxide from a 5-ml. microburette. Run a blank titration on a 
solution of alcohol (4 + 1) and adjust results accordingly. 

Calculations 

1 ml. 0.05N NaOH = 0.0072 gm. anhydrous sodium benzoate 

24 



If 1.2 ml. NaOH were required for titration: 

titer x normality x 0.0072 x dilution factor x 1,000,000 

p. p.m. = i 

0.05 x sample wt. 

= 1.2 x Q.Q5 x 0.0072 x lQ * 1,000,000 

0.05 x 100 

= 1.2 x 0.05 x 14,400 



25 



PICKLES 

SODIUM BENZOATE 



Procedure 

See section under "Mincemeat. " 



SODIUM CHLORIDE 

(Chromate Indicator Method) 

Principle 

An aliquot taken from a neutralized solution containing sodium chloride 
is titrated with a standardized solution of silver nitrate using potassium 
chromate as an indicator. 



Reagents 

1. Silver nitrate- 0.1N 

Dissolve 4.3 gm. reagent-grade silver nitrate in water and 
dilute to 250 ml. in a volumetric flask. Standardize against a 
solution containing 0.50 gm. sodium chloride (dried at 110° before 
weighing) per 100 ml. water. 

2. Potassium chromate 

Dissolve 5 gm. potassium chromate in water and dilute to 
100 ml. 



Procedure 

Take either a 20-gm. or 20-ml. sample. Add about 100 ml. water and 
neutralize to pH 5 to 7 with dilute sodium hydroxide. If a pH meter is not 
available, add methyl orange and enough alkali to change indicator from 
orange to yellow. Transfer the solution to 200-ml. volumetric flask, make 
up to volume, mix and filter through No. 4 Whatman paper. Pipette 50-ml. 
aliquot of filtrate into 150-ml. Erlenmeyer flask, add 1 ml. of potassium 
chromate solution and titrate with standard silver nitrate solution. The 
end point is the first permanent red color. 

Good lighting should be provided for the titration since the end point 
is extremely hard to detect with poor illumination. The analyst should 
satisfy himself that he is able to reproduce his results with a satisfactorily 
high degree of precision, since certain individuals are color blind to the 
particular color changes involved. 

26 



Calculations 

rl _ ml. AgNQ 3 * N AgN0 3 x equiv. wt. of NaCl x 100 

/C INclV^l — 777777^ r i 

1000 x wt. of sample 
= ml. AgN0 3 x N AgN0 3 x 58.45 x 100 
1000 x 5 (where dilution is 4) 

If the sample is taken by volume rather than by weight, report as 
percent w/v. 



References 

National Canners Association. Laboratory manual for the canning industry. 
Section 21, p. 13. National Canners Association, Washington, D. C. 
1954. 



27 



WINES AND CIDER 



PREPARING THE MATERIAL 



Before taking a sample of carbonated drinks for subsequent procedures 
it is good practice to pour the sample from one beaker to another numerous 
times. This helps to expel C0 2 and removes excess bubbles. If an abnormal 
amount of acetic acid is present, neutralize with IN NaOH. 



ALCOHOL 

(Pycnometer Method) 

Apparatus 

1. Constant-temperature water bath. 

2. Pycnometers, 50 ml. and 100 ml. 

3. Distillation apparatus with 500-ml. flask and 40-cm. condenser. 

Calibration 

Clean pycnometer and fill with distilled water. Place in water bath at 
temperature marked on pycnometer (usually 60° F.) and leave for 15 minutes. 
Adjust water level in flask until bottom of meniscus is exactly on graduation 
mark. With a piece of filter paper dry inside neck of pycnometer. Place back 
into bath for 10 minutes. Remove flask from water bath, dry, let stand for 
10 minutes at room temperature and weigh. Empty pycnometer, rinse with 
acetone and dry with air stream. Let flask come to room temperature, stopper 
and weigh. 

Procedure 

Fill a dry 100-ml. pycnometer with sample and adjust to temperature 
specified. Transfer contents to the distillation flask. Rinse pycnometer 
three times, using a total of 50 ml. cold water, adding the rinsing to the 
flask. If foaming is expected, add a small amount of tannin. Complete 
distillation connections and distill into pycnometer flask until a volume of 
about 95-98 ml. has been collected. Remove pycnometer and place in water 
bath at constant temperature specified on pycnometer. After 15 minutes 
dilute exactly to the mark, using water at the same temperature. Dry inside 
neck and outside of pycnometer and then weigh. 

Calculations 

f ,. .,, wt. pycnometer with distillate - wt. empty pycnometer 

sp.gr. of distillate = — —. r / r/ 

wt. pycnometer with water - wt. empty pycnometer 

From an alcohol table for 60° F.find the corresponding alcohol content 

(% by volume). 

28 



ALCOHOL 

(By Distillation and Hydrometer) 

Principle 

A measured volume of sample is distilled and the distillate diluted to a 
definite volume, usually the original volume. The alcohol content of the 
distillate is determined by means of a hydrometer. 

Procedure 

Pipette 100-ml. sample into the distillation flask and add 50 ml. water. 
Add a small amount of tannin if foaming occurs. (Note: A small amount of 
added Antifoam "A" (Dow Corning) prevents foaming.) If an abnormal 
quantity of acetic acid is present, neutralize exactly with IN NaOH. Place 
a 250-ml. Erlenmeyer flask in position to collect the distillate. With low 
heat distill about 90 to 95 ml. into the flask. Remove the flask, transfer 
distillate to a 200-ml. volumetric flask, cool to required temperature and 
make up to volume with water. 

Note the temperature on the stem of the hydrometer. Cool the distillate 
to exactly this temperature in an ice bath. Fill the hydrometer cylinder 
with distillate, check temperature and insert hydrometer. Read the percent 
alcohol at the bottom of the meniscus, that is, at the general level of the 
liquid. Multiply the result by the dilution factor to obtain the percentage of 
alcohol. 

A specific gravity hydrometer may be used instead of one reading in 
percent alcohol. Tables are available to convert specific gravity of alcohol — 
water mixtures to percent alcohol by volume. 



TOTAL ACIDITY 

Principle 

Total acidity is determined by direct titration with 0.1N NaOH to pH 
8.1, using a pH meter. 

Procedure 

Weigh 10-gm. sample into a 250-ml. beaker. Add 100 ml. water and 
bring quickly to boil to expel C0 2 . Do not continue boiling because volatile 
acids may be lost. Cool the sample and titrate to pH 8.1 with 0.1N NaOH, 
using a pH meter. Record volume of NaOH required and calculate the total 
acidity as percent of the predominant acid. 

Calculations 

1 ml. 0.1N NaOH = 0.0075 gm. tartaric acid 

29 



If 5.8 ml. 0.1N NaOH were required for titration: 
„, , . , 1 x equiv. wt. of acid x normality of NaOH x titer 

% total acid = zrrr- — r : - 

10 wt. ol sample 

1 x 75.0 x Q.l x 5.8 
"10 10 

Note: See page 3 for equivalent weights of acids. 



TANNIN AND COLORING MATTER 

Principle 

A de-alcoholized sample is titrated with standard potassium perman- 
ganate using indigo solution as an indicator. The volume of permanganate 
solution required minus that required to oxidize a similar aliquot from which 
the tannin and coloring matter were removed is the volume of permanganate 
required to oxidize the tannin. 

Reagents 

1. Potassium permanganate— 0. IN 

Dissolve 3.160 gm. in about 200 ml. water. Transfer to 1,000-ml. 
volumetric flask and makeup to volume with water at 20°. Standardize 
with sodium oxalate as on page 46. 

2. Indigo solution 

Dissolve 3 gm. sodium indigotin disulfonate in 200 ml. water 
by heating, cool, add slowly 25 ml. cone. H 2 S0 4 . Transfer to 500-ml. 
volumetric flask and dilute to mark with water. 

3. Purified boneblack 

Boil 200 gm. powdered boneblack with two successive portions 
of HC1 (1 + 3). Filter through hard filter paper, wash with boiling 
water until chloride-free (test with AgN0 3 ). Place in flask and keep 
covered with water. If acid-washed charcoal is available, this step 
can be eliminated. 

Procedure 

Pipette 100 ml. wine or cider into 400-ml. beaker. Add about 25 ml. 
water and remove alcohol by evaporating on a hot plate to about 75 ml. 
Cool, transfer to 100-ml. volumetric flask, rinsing the beaker several times. 
Cool and make up to volume. 

Pipette 10 ml. of the de-alcoholized sample into a 2,000-ml. porcelain 
evaporating dish. Add 1,000 ml. water and exactly 20 ml. indigo solution 
(reagent 2). Add the standard KMn0 4 solution from a burette 1 ml. at a time 
until the blue color changes to green, then add a few drops at a time until 
the color becomes golden yellow. Designate number of ml. of KMn0 4 solution 



n >> 

as a. 



30 



Transfer remaining de-alcoholized sample from the volumetric flask to a 
250-ml. Erlenmeyer flask. Add about 1 gm. prepared boneblack and after 10 
minutes, with occasional shaking, filter through No. 41H or No. 5 paper. 
The carbon adsorbs tannin, other nontannins and anthocyanins. The filtrate 
must be crystal clear. 

Pipette 10 ml. of decolorized sample into a 2,000-ml. porcelain evap- 
orating dish. Add 1,000 ml. water and exactly 20 ml. indigo solution. Titrate 
with the KMn0 4 solution as directed above. Designate ml. KMn0 4 as "b." 

Calculations 

1 ml. 0.1N KMn0 4 = 0.00416 gm. tannin and coloring matter. 

or . • i i • «,•.•.-■ (a - b) x normality of KMnQ 4 x 4.16 
7c tannin and coloring matter = - - 1 ±±. 

wt. of sample titrated 

References 

Cruess, W. V., M. A. Joslyn and L. G. Saywell. Laboratory examination of 
wines and other fermented products, pp. 66-69. Avi Publishing Co. Inc., 
New York, N.Y. 1934. 



TOTAL AND REDUCING SUGARS 

(Total Sugar — Dry Wines and Cider) 

Reagents 

See "Jams, Jellies and Marmalades.' ' 

Preparing the Sample 

Weigh 200-gm. sample into 400-ml. beaker, neutralize with IN NaOH to 
pH 8.1 on meter and evaporate to about 100 ml. Cool, transfer to 200-ml. 
volumetric flask and make up to volume at 20°. Transfer to 400-ml. beaker, 
add about 1 gm. decolorizing charcoal, shake, and let stand for 10 minutes. 
If necessary add one teaspoon of Hyflo Super-eel or similar filter aid, shake 
and filter through 41H paper. The filtrate must be clear and if so may be 
used directly in the "Standard Method" titration as under "Jams, Jellies and 
Marmalades. " This applies where it can be safely assumed that the sugar 
present is all reducing sugar. 

Calculations 

factor (from Table 3) x 100 



% sugar = 



titer 1000 

31 



(Total and Reducing Sugar — Sweet Wines and Cider) 
Preparing the Sample 

Weigh 25-gm. sample into 400-ml. beaker. Add about 150 ml. water and 
neutralize to pH 7.5-8.0 with IN NaOH. Evaporate to about 100 ml. (20-25 
minutes' boiling). Cool, transfer to 250-ml. volumetric flask, add 100-200 
ml. water and 2 ml. lead acetate solution. Shake, and let stand for 10 
minutes. Add the necessary amount of potassium oxalate (see "Jams, Jellies 
and Marmalades"), bring up to volume with water and filter through 41H or 
No. 5 paper. Test filtrate with small amount of potassium oxalate to deter- 
mine if lead is absent. If filtrate is clear, follow the procedure for "Total 
and Reducing Sugars/' under "Fruit Juices," using 50-ml. aliquots. 

Calculations 

See "Total and Reducing Sugars," under "Fruit Juices." 

References 

Cruess, W.V., M.A. Joslyn and L. G. Saywell. Laboratory examination of 
wines and other fermented products, pp. 52-58. Avi Publishing Co. 
Inc., New York, N.Y. 1934. 



IRON 

(Colorimetric Method) 
Wet Ashing 

Reagents 

1. Nitric acid — concentrated 

2. Perchloric acid - 72% HC10 4 

Procedure 

Pipette 25 ml. of thoroughly degassed sample into 125-ml. Erlenme/er 
flask and boil down to a thick syrup, slightly charred. Add 20-25 ml. HN0 3 
and 2 ml. HC10 4 to flask and heat gently in fume cabinet until initial 
reaction begins. This is indicated by vigorous boiling with evolution of 
brown nitrogen tetroxide. 

After reaction has subsided, again heat contents of the flask to slow 
boiling and continue boiling until all HN0 3 is driven off as evidenced by 
evolution of fumes of HC10 4 . When cooled, the residue should be colorless 
or at the most a pale yellow. If not, add small amounts of HN0 3 and HC10 4 
and heat further. After contents are completely ashed and cooled, transfer 
to 100-ml. volumetric flask and make up to volume. 

Preparation of Standard Curve 

Reagents 

1. Glass-distilled water 

32 



2. Acetic acid-2M 

Dilute 120 gm. acetic acid to 1 liter with water. 

3. Ammonium citrate solution— 1% 

Dissolve 1 gm. ammonium citrate in water and dilute to 100 ml. 

4. Bromophenol blue indicator— 0.04% 

Dissolve 0.1 gm. bromophenol blue in 3 ml. 0.05N NaOH, 
transfer to a volumetric flask and dilute to 250 ml. with water. 

5. Buffer solutions 

(a) pH 3.5 - Mix 6.4 ml. 2M sodium acetate with 93.6 ml. 2M 
acetic acid and dilute to 1 liter. 

(b) pH 4.5 - Mix 43 ml. 2M sodium acetate with 57 ml. 2M 
acetic acid and dilute to 1 liter. 

6. Hydroquinone solution— \% 

Dissolve 1 gm. hydroquinone in 100 ml. buffer solution (pH 4.5). 
Store in refrigerator and discard as soon as any color develops. 

7. o-Phenanthroline solution 

Dissolve 1 gm. o-phenanthroline monohydrate in water and 
dilute to 400 ml. 

8. Sodium acetate solution— 2M 

Dissolve 272 gm. NaC2H 3 2 .3H 2 in water dilute to 1 liter. 

9. Standard iron solution — No. 1 — 1 mg. Fe/ml. 

Dissolve 1 gm. electrolytic iron in 50 ml. 10% H 2 S0 4 . Cool 
and dilute to 1 liter with water. 

10. Standard iron solution— No. 2 

Pipette 5 ml. of standard iron solution No. 1 into 500-ml. 

volumetric flask and make up to volume with water. One ml. of 

this solution contains 0.01 mg. Fe. (10 meg.) 
Pipette 2-, 4-, 6-, 8- and 10-ml. aliquots standard iron solution No. 2 
into 25-ml. volumetric flasks and the same amounts into test tubes. Add 5 
drops bromophenol blue indicator to aliquots in the test tubes and add 
sodium acetate solution until color matches that of equal volume of buffer 
solution of pH 3.5 containing same quantity of indicator. Add 1 ml. of 
hydroquinone solution and 2 ml., o-phenanthroline solution to the aliquots 
in the volumetric flasks. Adjust pH of the contents to 3.5 by adding same 
volume of sodium acetate solution as found necessary for aliquot in test 
tubes. Prepare a blank according to above procedure but omitting standard 
iron solution No. 2. Make all 25-ml. volumetric flasks up to volume with 
water, mix thoroughly and plot optical density against concentration on 
graph paper at wave length of 510 m/x. 

Procedure 

Pipette 10 ml. of digested sample into 25-ml. volumetric flask and same 
amount into a test tube. Add 5 drops bromophenol indicator to aliquot in 

33 



test tube and titrate with sodium acetate solution until color matches that 
of equal volume of buffer solution of pH 3.5 containing the same quantity 
of indicator. 

Add 1 ml. hydroquinone solution and 2 ml. o-phenanthroline solution to 
aliquot in volumetric flask. Adjust pH of contents to 3.5 by adding same 
volume of sodium acetate solution as was found necessary for aliquots in 
test tube. If turbidity develops upon adjustment of pH of aliquot in test 
tube, add 1 ml. NH 4 citrate solution to volumetric flask before adding the 
sodium acetate solution. 

Prepare blank according to the above procedure. Make all flasks up to 
volume with water andmix thoroughly. Let stand fori hour to assure complete 
color development. Take colorimeter readings and plot optical density on 
the same graph paper as the standard curve. 

Calculations 

p. p.m. Fe = meg. Fe from curve divided by 2.5. 

References 

Association of Official Agricultural Chemists. Official methods of analysis. 
8th ed. Washington, D.C. 1955. 



TOTAL VOLATILE ACIDITY 

Principle 

Volatile acids are steam-distilled from the sample using a Hortvet type 
distillation apparatus, and titrated with standard sodium hydroxide. The 
acids are calculated as acetic acid. 

Procedure 

Boil about 200 ml. of water in the outer heating flask. Most flasks are 
provided with an outside vent tube. The water may be boiled in the outer 
flask with the vent tube open to remove the dissolved C0 2 , as well as to 
replace with steam the air in the flask surrounding the inner tube. Apply 
heat gently and turn on cold water through condenser. 

With a pipette, introduce a 20-ml. sample into inner tube and connect 
at once to the condenser. Increase the heat and bring the water in flask to 
vigorous boiling, having the pinch cock on side of the tube open. When the 
water is boiling vigorously, close the pinch cock. Steam passes through the 
20-ml. sample, carrying the volatile acid into the condenser. Collect the 
condensate in a 250-ml. Erlenmeyer flask. Continue distillation until 100 
ml. is collected. 

Transfer the 100 ml. of distillate to 250-ml. beaker. Add about 50 to 
100 ml. of water and titrate with 0.1N NaOH to pH 8.1. 

34 



Calculations 

1 ml. 0.1N NaOH = 0.006 gm. acetic acid 

Note: 

To determine whether all the volatile acids have been extracted, 50 ml. 
of distillate is first collected in one flask and titrated. An additional 10-ml. 
portion is then collected in a second flask and added to the distillate in the 
first flask and titrated. Further 10-ml. portions are distilled over until an 
additional 10-ml. portion does not change the titration by more than one or 
two drops. Generally only 80 ml. of distillate is required. 

References 

Cruess, W.V., M. A. Joslyn and L. G. Saywell. Laboratory examination of 
wines and other fermented products, pp. 33-44. Avi Publishing Co. 
Inc., New York, N.Y. 1934. 

VOLATILE ACIDITY 

(Exclusive of S0 2 ) 

Principle 

After removal of the free sulfur dioxide by addition of barium hydroxide, 
the volatile acids are steam-distilled from the sample and titrated with 
standard sodium hydroxide. 

Procedure 

Pipette 50-ml. sample into 250-ml. beaker, and add enough clear 
saturated Ba(OH) 2 solution to bring mixture to pH 8.1. Allow to stand 30 
minutes and maintain at pH 8.1 by adding more Ba(OH) 2 if necessary. 
Transfer to 100-ml. volumetric flask, dilute to volume and filter immediately 
through No. 2 Whatman paper. Pipette 20 ml. of filtrate into inner tube of 
volatile acidity distillation flask and add 1 ml. of H 2 S0 4 (1 + 3). Place 
150 ml. recently boiled hot water in outer flask and distill 100 ml. Using 
pH meter, titrate with 0.1N NaOH to pH 8.1. 

1 ml. 0.1N NaOH = 0.006 gm. acetic acid 

References 

Association of Official Agricultural Chemists. Official methods of analysis. 
8th ed. p. 189. Washington, D.C. 1955. 



EXTRACT 

The "extract" of wine and cider represents the alcohol-free soluble 
solids present and consists mainly of tartaric acid, potassium bitartrate, 
malic acid, protein, coloring matter, sugar and gums. 

35 



One method of determining the extract is to de-alcoholize the sample 
by boiling, dilute to the original volume and determine specific gravity 
with a Brix or Balling hydrometer. The other method is to evaporate a 
measured volume of sample to dryness and weigh the extract. 

(Hydrometer Method) 

Procedure 

Pipette 100-ml. sample into 400-ml. beaker. Add 50 ml. water and 
evaporate slowly to a volume of about 50 ml. Avoid loss by spattering. 
Transfer to 100-ml. volumetric flask and rinse the beaker with water. Add 
the washings to the flask. Cool and dilute to mark. Transfer to a cylinder 
and insert a Brix or Balling hydrometer. The reading gives the grams of 
extract per 100 ml. of original sample. (For approximate purposes the 
refractometer reading gives a satisfactory result.) 

(Oven Method) 

Procedure 

Place empty evaporating dishes in oven at 100° for 1 hour. Transfer to 
desiccator, cool and weigh. Pipette 50-ml. sample into evaporating dish and 
heat on water bath until the liquid has evaporated to a viscous consistency. 
Place dishes in vacuum oven at 70° and 26 to 28 inches vacuum for 8 hours. 
Place in desiccator, cool and weigh. 

Calculations 

wt. of extract * 100 



% extract = 



wt. of sample 



36 



SAUERKRAUT 

TOTAL ACIDITY 

Procedure 

From the liquid portion of the product weigh 5 gm. into 250-ml. beaker. 
Add 100 ml. water, boil for a few minutes to drive off C0 2 , cool and titrate 
with pH meter to pH 8.1, using 0.1N NaOH. 

Calculations 

~ , . . , titer x N x equiv. wt. x 100 

% lactic acid = — * — ; : 

1,000 x wt. of sample 

If 7.2 ml. 0.1N NaOH were required for titration: 

m . . ., 7.2 x 0.1 x 90.08 x 100 

% lactic acid = ., „~~ = 

1,000 x 5 

= 1.3 

pH 

Procedure 

Adjust pH meter with pH 4.0 buffer solution. 

Pour 50 to 75 ml. of juice into 100-ml. beaker and take pH reading. 



FRUIT PRESERVED IN SULFUR DIOXIDE 

SULFUR DIOXIDE 

(Official Method) 

Preparing the Material 

Place portion of material for analysis free from pits into Waring blendor 
jar. Blend just long enough to mix into a slurry. Place portion into an 
airtight container until ready for analysis. 

Procedure 

Using 25 gm. blended sample, follow the procedure for "Jams, Jellies 
and Marmalades. " 

(Control Method) 

Principle 

The sulfur dioxide solution is titrated directly against 0.05N iodine 
solution using starch as indicator. This control method is useful for de- 
termining the amount of sulfur dioxide in stock solutions or for estimating 
the sulfur dioxide content of fruit pulp preserved in this manner. 

37 



Reagents 

1. Iodine solution— 0.05N 

Dissolve 6.346 gm. iodine in a solution of 12 gm. potassium 
iodide in 100 ml. water and dilute to 1 liter. 

2. Starch solution 

Mix 0.5 gm. soluble starch with a little cold water (about 15 
ml.), pour into 100 ml. hot water and boil 1 to 2 minutes. Add enough 
NaCl to saturate, and store in a refrigerator, where it keeps for 
several weeks. 

Procedure 

With a 100-ml. pipette (inverted), remove about 100 ml. solution through 
the bunghole of the barrel. If the solution is not clear, filter through No. 4 
Whatman paper into a 400-ml. beaker. Transfer the filtered solution to a 
50-ml. burette as quickly as possible. 

Pipette 10 ml. 0.05N iodine solution into a 500-ml. Erlenmeyer flask 
containing about 100 ml. water. Add 1 ml. starch solution. Titrate the 
S0 2 solution from the burette into the flask containing the iodine solution, 
rotating the flask frequently to keep the solution well mixed. When the 
color of the iodine solution becomes purple, add the solution from the 
burette dropwise, stopping at the point where one drop causes all color to 
disappear from the iodine solution. 

Calculations 

1 ml. 0.05N iodine reacts with 0.0016 gm. S0 2 

Qn _ ml. iodine * normality of iodine x 3.2 

% o(J 2 — i — rvA i ' : 1 

ml. bu 2 solution required 

p.p.m. S0 2 = % S0 2 x 10,000 

References 

Atkinson, F.E., and C.C. Strachan. Preservation of fruits with sulfur 
dioxide in British Columbia. Fruit Prod. J. 21: 5-8; 43-45; 60; 72-74; 
110-112; 141-144; 153. 1941. 

Wiegand, E. H. Process for the manufacture of maraschino cherries. Western 
Canner and Packer 29: 33-34. 1937. 



38 



MISCELLANEOUS PROCEDURES 

CALCIUM 

(Official Method) 

Principle 

Calcium is precipitated as calcium oxalate. The precipitate is dissolved 
in hot dilute sulfuric acid and titrated with standard potassium permanganate. 

Ashing 

Weigh duplicate 25-gm. blended samples into glazed procelain dishes. 
Evaporate to dryness on water bath or in forced-air oven at 100° and ash at 
low red heat (not to exceed 525°) until free of carbon particles. 

Reagents 

1. Methyl orange-0.05% 

Dissolve 0.05 gm. methyl orange in water and dilute to 100 ml. 

2. Oxalic acid-2.5% 

Dissolve 12.5 gm. oxalic acid in water and dilute to 500 ml. 

3. Sodium acetate— 20% 

Dissolve 100 gm. sodium acetate in water and dilute to 500 ml. 

4. Saturated ammonium oxalate solution. To 12.0 gm. (NH4) 2 C 2 04. H 2 add 

200 ml. of water. 

Procedure 

Dissolve the ash obtained above in 50 ml. of HC1 (1 + 4) and heat for 
a few minutes. Be sure residue is acid. Filter through 15-cm. diameter No. 2 
Whatman paper and wash thoroughly. Collect the washings in a 200-ml. 
volumetric flask. Make up to volume at 20°. To this filtrate or an aliquot, 
add 2 drops of methyl orange, and then ammonium hydroxide (1+ 4) drop by 
drop, until the solution is just alkali. Add dilute HC1 (1 + 4) drop by drop 
until the solution is just acid. (When solution is cold and acid to the 
indicator, all the calcium phosphate is in solution. A small amount of 
phosphate or iron may remain undissolved at this point, but goes into 
solution when 0.5N acid is added.) 

When the solution is just acid, add 10 ml. of 0.5N HC1 and 10 ml. 
2.5% oxalic acid. Heat the solution to the boiling point. Add 10 ml. of 20% 
solution of sodium acetate with constant stirring. Boil gently for 10 minutes. 
Add a few drops of saturated ammonium oxalate solution to make sure that 
all the calcium is precipitated. Hold overnight at 32 to 40° F. 

Filter through 11-cm. diameter No. 40 Whatman paper into a beaker. 
Wash the precipitate free of chlorides with cold water (test with AgN0 3 ). 
Wash the precipitate into a 400-ml. beaker using hot water from wash bottle. 

39 



Keep the filter paper to add later in the titration. Make up to about 200 
ml. with water, add 5 ml. cone. H 2 S0 4 and heat to 70 to 80° C. Titrate hot 
with 0.1N KMn0 4 almost to completion (slight pink color). Add the filter 
paper in strips and complete titration to the first permanent pink color. 

Calculations 

1 ml. 0.1N KMn0 4 = 0.002 gm. calcium 

References 

Association of Official Agricultural Chemists. Official methods of analysis. 

8th ed. p. 378. Washington, D.C. 1955. 
Snell, D. F., and C.T. Snell. Colorimetric methods of analysis. Vol. II. 

3rd ed. D. Van Nostrand Co., New York. 1949. 

TANNIN AND COLORING MATTER 

(Fruits and Fruit Products) 

Principle 

In a neutral solution, tannin and coloring matter react with permanganate 
and are measured by titration, using indigo solution as an indicator. As 
these solutions contain other oxidizable matter besides tannin, it is nec- 
essary to separate these using charcoal and titrating a second time to 
determine the quantity of permanganate actually required by the tannin 
present. 

Reagents 

See "Tannin and Coloring Matter, " under "Wines and Cider." 

Procedure 

For light-colored products such as peach, apple and pear, use a blended 
50-gm. sample; for deeper-colored products, use 25 gm. 

Transfer the sample to 600-ml. beaker, add 300 ml. of water and boil 
gently for 1 hour, replacing the water lost by evaporation.Cool, transfer to 500-ml. 
volumetric flask and dilute to mark. Mix thoroughly and filter through No. 4 
Whatman paper. 

Pipette 400 ml. of filtrate into 600-ml. beaker, add 0.3 gm. powdered 
CaC0 3 and heat to boiling. Cool, transfer to 500-ml. volumetric flask and 
make up to volume. Mix thoroughly, and filter through No. 5 Whatman paper, 
refiltering if necessary until brilliantly clear. 

Pipette 200 ml. filtrate into 2-liter procelain dish, add about 800 ml. 
of water and exactly 20 ml. of the indigo solution. Add standard KMn0 4 
solution 1 ml. at a time, stirring vigorously until the blue color changes to 
green, then add a few drops at a time until the color becomes a golden 
yellow. Designate the ml. of KMn0 4 used as "a." 

40 



To the remaining filtrate add 1 gm. carbon and shake intermittently 
for 10 minutes. Filter through No. 5 Whatman paper, refiltering if necessary 
until clear. Pipette 200 ml. filtrate into the porcelain dish and add 800 ml. 
water and exactly 20 ml. of the indigo solution. Titrate with standard KMn0 4 
in the manner described above. Designate the ml. of KMn0 4 solution required 



as 



'b." 



Calculations 

1 ml. 0.1N KMn0 4 = 0.0035 gm. tannin 

(a - b) = ml. KMn0 4 solution required for oxidation of tannin 

_, . (a - b) x normality of KMn0 4 x 3.5 

% tannin = — 

wt. of sample titrated 

References 

Hartman, B. E. The polybasic acids of fruits and fruit products. Tannin 
and coloring matter. J. Assoc. Offic. Agr. Chemists 26: 452-462. 1943. 

Strachan, C.C., A. W. Moyls, F.E. Atkinson and J. E. Britton. Chemical 
composition and nutritive value of British Columbia tree fruits. Canada 
Dept. Agr. Pub. 862. 1951. 



ENZYME TESTS FOR ADEQUACY OF BLANCHING IN FROZEN 

VEGETABLES 

Principle 

This method is based upon measurement of the rate of color develop- 
ment in a guaiacol - hydrogen peroxide substrate under the catalytic influ- 
ence of the enzyme present in the tissue. The reaction is brought about 
through the formation of an active peroxidase-peroxide complex, which 
oxidizes the colorless- guaiacol directly to an orange-brown end product. 

Reagents 

1. Guaiacol solution— 1% 

Dissolve 1 gm. or 0.9 ml. guaiacol in 50 ml. ethyl alcohol and 
add 50 ml. water. 

2. Hydrogen peroxide— 1% 

Dilute 1 part 3% H 2 2 (free from preservatives) with 2 parts water. 

Note: Glass dropping bottles of 100-ml. capacity are ideal 
containers. The reagents should be protected from light and stored 
in a refrigerator. 

Testing reagents. The effectiveness of the reagents is deter- 
mined by carrying out tests on two small pieces of fresh vegetables, 
one of which is boiled for 10 minutes and cooled. The fresh material 
should give a positive test, the heated one a negative test. 

41 



Preparing the Material 

Select representative material from portions that were heated least in 
the blanching, i.e., the central portions of the thickest pieces. Use a 
stainless steel cutting knife. 

For spinach, chard or similar leafy material, select a number of leaves 
and take the inch midrib portion beginning at the base of the leafy portion. 

For asparagus spears, cut off and discard 3/4 inch from the butt end, 
then split the spears lengthwise. 

For broccoli and cauliflower, split the stalk and head lengthwise. 

For peas and other seed vegetables, cut each seed in half. 

For string beans, cut 1/4- to 1/2-inch cross sections from a number 
of beans and split these cross sections lengthwise. 

Procedure 

Place the prepared material on a white porcelain saucer or evaporating 
dish. Add enough guaiacol solution to wet all of the cut surfaces, then 
immediately add a similar amount of hydrogen peroxide solution. At the 
end of 3 minutes note whether a reddish-brown color has developed. If 
none is observed the test for peroxidase is negative. Neglect any color 
that may develop after 3 minutes. The reactions, as read at the end of 3 
minutes, are graded as follows: 

negative — no color 
trace — reddish-brown specks 

faint — up to 25% of the material colored 

heavy — material a solid reddish-brown color 
References 

Atkinson, F.E., C.C. Strachan and A. W. Moyls. B.C. Processor's Hand- 
book. Fruit and Vegetable Processing Laboratory, Canada Dept. Agr. 
Experimental Farm, Summerland, B.C. Sept. 1947. 
Joslyn, M. A. Report on peroxidase in frozen vegetables. J. Assoc. Offic. 
Agr. Chemists 36: 161-178. 1953. 



CRUDE FAT OR ETHER EXTRACT 

(For Fruit and Vegetable Products) 

Principle 

Fat-soluble material is extracted from an oven-dried sample using a 
Soxhlet extraction apparatus. The ether is evaporated and the remaining 
material weighed. 

Procedure 

Weigh 50-gm. blended sample into a 250-ml. beaker. Add about 75 ml. 
water and about 5 gm. asbestos. Mix and filter through No. 4 Whatman 

42 



filter paper. If globules of fat are present on the water layer, decant liquid 
into a separatory funnel and extract with several small portions of ether. 
If no fat is observed, liquid layer may be discarded. Keep the ether extract 
and combine it with sample before drying. 

Place residue and filter paper in a thin aluminum foil dish and dry 
at 100° until moisture is removed, usually overnight. 

Remove from oven and when cool, cut the dish and contents into small 
pieces and transfer directly into Soxhlet extraction thimble. Extract in the 
Soxhlet apparatus with anhydrous ether for at least 16 hours. 

Remove thimble from the apparatus and distill off most of the ether by 
allowing it to collect in the Soxhlet tube and pouring it off when the tube is 
nearly full. When the ether has reached a small volume, pour it into a small 
beaker through a small funnel containing a plug of cotton. Rinse the flask 
and filter thoroughly, using several small portions of ether. 

Evaporate the ether on a steam bath at low heat, preferably under 
a current of air. Dry at 100° for 1 hour, cool and weigh. 

Calculations 

wt. of fat-soluble material x 100 



% crude fat = 



wt. of sample 



References 

National Canners Association. Laboratory manual for the canning industry. 
Section 20, p. 32. National Canners Association, Washington, D. C. 
1954. 



ESTIMATION OF CALORIE CONTENT OF DIET FOODS 

(Dietetic Fruit Spreads) 

Take refractometer reading of sample at 20°. Multiply the reading by 4 
to estimate number of calories per 100 gm. 
If soluble solids reading is 21%: 
Calories per 100 gm. = 21.0 x 4 

= 84. 



43 



APPENDIX 

STANDARD SOLUTIONS 

ACIDS 



Hydrochloric Acid-O.IN (3.646 gm. per liter) 

Use cone. HC1 (strength usually stated on bottle). 

3.646 x 100 era. - rt _ __ TT/ ^, . _ ,. . „ , XT . 
r=-r sL_ of 37.3% HC1 gives 1 liter of 0.1N solution 

O/aO 

Sp. gr. of cone. HC1 = about 1.19 
Therefore volume of cone. HC1 required: 

3.646 x 100 . , ,. . A ... . . 
= o.z ml. per liter lor 0.1JN solution 

Standardize against: 

Standard 0.1N NaOH (titrate to pH 8.1) 



Succinic Acid-O.IN H 2 C 4 H 4 4 (5.9023 gm. per liter) 

Dry 5 to 6 gm. pure succinic acid in open weighing bottle at 105° for 
about 10 hours; cool and store in desiccator. Weigh 2.9511 gm., transfer to 
400-ml. beaker and dissolve in 150 to 200 ml. of water. Pour the solution 
into 500-ml. volumetric flask, rinsing out the beaker several times to insure 
complete transfer of the acid. Dilute to exactly 500 ml. and mix thoroughly. 
This prepares an exact 0.1N solution. 

Sulfuric Acid-O.IN solution (4.904 gm. per liter) 

Pour 3 ml. of cone. H 2 S0 4 carefully into 10-12 ml. of water. Cool, mix 
thoroughly and dilute to 1 liter. Standardize by titration against standard 
NaOH or KOH to the phenolphthalein end point or to pH 8.1 with a pH 
meter. 



Oxalic Acid-normal (63.023 gm. H 2 C 2 4 .2H 2 per liter) 

Decinormal or less concentrated solutions are unstable and should be 
prepared fresh when needed. More concentrated solutions may deposit some 
of the acid when cooled to low temperatures but they are fairly stable at 
room temperature when protected from light. 

44 



BASES 

Sodium Hydroxide-normal (40.005 gra. per liter) 

Dissolve 42 gm. C.P. sodium hydroxide pellets in water and dilute to 1 
liter in a volumetric flask. 

Standardization of sodium hydroxide 

1. For procedures using pH 8.1 as the end point: against weighed portions 
of succinic acid. 

Dry succinic acid crystals at 105° for 8 hours and cool in desiccator. 
Take suitable portions of succinic acid (about 0.1 gm. for 0.1N NaOH and 
0.05 gm. for 0.05N NaOH) and weigh accurately on an analytical balance. 
Dissolve in about 150 ml. water in 250-ml. beakers. Titrate in duplicate 
with NaOH solution to pH 8.1. 

Calculations 

AT ,. . AT ~ TT wt. of succinic acid x 1,000 

Normality of NaOH = — , . T ^ TT : - t ; , 

mi. NaUH x equiv. wt. ot acid 

If 0.1047 gm. succinic acid required 17.1 ml. NaOH: 

tvt i- r at nu 0.1047 x 1,000 

Normality of NaOH ^ 171x59>( ; 3 

2. For procedures using bromophenol blue as indicator; against standard 
H 2 S0 4 . 

Standardization of H 2 S0 4 against Na 2 C0 3 

Heat Na 2 C0 3 at 105° for 8 hours. Weigh out exactly 1.3250 gm., dissolve 
and make up to 250 ml. This makes exactly 0. IN Na 2 C0 3 solution. 

Pipette 5 ml. H 2 S0 4 into 125-ml. Erlenmeyer flask. Add about 25 ml. 
water and 4 drops bromophenol blue. Titrate Na 2 C0 3 from burette to a blue 
end point or pH 4.1, using a pH meter. 

Standardization of NaOH against H 2 SO A 

Pipette 5 ml. standardized 0.1N H 2 S0 4 into 125-ml. Erlenmeyer flask. 
Add about 25 ml. water and 4 drops bromophenol blue indicator. Titrate 
NaOH from burette to color end point, or pH 4.1 using a pH meter. 

For 0.05N NaOH use 2 ml. H 2 S0 4 . 



OXIDIZING AND REDUCING SOLUTIONS 

Potassium Dichromate-O.IN (4.9037 gm. per liter) 

Dry crystals at 120 to 140° for 2 to 4 hours. Cool in a desiccator and 

weigh 5.0 gm. to the nearest milligram. Dissolve in about 200 ml. water and 

transfer to 1-liter volumetric flask, dilute to volume and mix thoroughly." 

AT ,. wt. of potassium dichromate 

lNormalitv - * 

y 49.037 

45 



Potassium Permanganate-O.IN (3.1606 gm. per liter) 

Dissolve 3.3 gm. of dry KMn0 4 in about 200 ml. water and transfer to a 
1-liter flask. Make up to volume at 20°. 

Standardization with sodium oxalate 

Weigh out accurately three 0.25— to 0.30-gm. samples of sodium oxalate 
having an assay value of 99.95%, transfer each portion to a 600-ml. beaker, 
using 250 ml. dilute sulfuric acid (5 + 95). Stir until the oxalate has dis- 
solved, then add rapidly from a burette about 95% of the amount of perman- 
ganate needed for complete oxidation of the sample. Allow the solution to 
stand until the permanganate is decolorized, then heat to 55 to 60°. Complete 
the titration at this temperature, stirring gently and allowing each drop to 
become decolorized before adding the next; titrate to first permanent pink. 

AT ,. P T ,,„ ~ gm. sodium oxalate x 1,000 

Normality of KMn0 4 = § — ; . ■ . g 

7 ml. of KMn0 4 x 67.0 

Sodium Thiosulfate-O.IN (24.8192 gm. Na 2 S 2 8 . 5H,0 per liter) 

Weigh 25.0 gm., dissolve in 200 ml. water, transfer to 1-liter flask and 
make up to volume. Mix the solution thoroughly, allow it to stand for a few 
days, and then siphon off the clear liquid. The solution is standardized 
indirectly with potassium dichromate. 

Standardization with potassium dichromate 

Accurately weigh 0.20- to 0.23-gm. K 2 Cr 2 7 (dried 2 hours at 105°). 

Transfer to 250-ml. beaker using about 150 ml. water. Add 2 gm. potassium 

iodide and mix. Add 20 ml. IN HC1, swirl, and let stand for 10 minutes. 

Start titrating with the sodium thiosulfate from burette, adding about 80% 

of the required amount. Add 1 ml. starch and complete titration to point 

where solution changes from blue-green to light green. 

M ,- f gm. K 2 Cr 2 Q 7 x 1000 

Normality = —. — — — p— ^ tz \ n r7 

7 ml. Na 2 S 2 3 x 49.037 

lodine-O.lN (12.693 gm. per liter) 

Dissolve 13.5 gm. pure resublimed iodine in a solution of 24 gm. 
potassium iodide in 200 ml. of H 2 and dilute to 1 liter. The solution is 
standardized by titrating against a known volume of standard thiosulfate, 
with a few drops of starch solution as indicator. 



INDICATORS 

Phenolphthalein-pH range 8.3 to 10 

Dissolve 1 gm. in 100 ml. neutral ethyl alcohol and water. Use 1 drop 
per 100 ml. solution. 

46 



Methyl red— pH range 4.4 to 6.0 

Dissolve 1 gm. in 100 ml. 95^ ethyl alcohol. This indicator is easily 
reduced with loss of color, and readings must be made shortly after it is 
added to the solution. 

Methyl orange-pH range 2.9 to 4.0 

Dissolve 0.5 gm. in 1000 ml. water. 

Bromophenol blue— pH range 3.0 to 4.6 

Dissolve 0.1 gm. in 25 ml. water and dilute to 100 ml. with water. 

Starch solution-0.5% 

Dissolve 0.5 gm. soluble starch in about 15 ml. cold water and pour 
into 100 ml. hot water. Boil 1 to 2 minutes. 

Cleaning solution 

Sodium or potassium dichromate (commercial) 40 gm. 

Water 150 ml. 

Dissolve with a little heat if necessary, then cool to room temperature 
and add slowly 230 ml. cone, sulfuric acid (tech.). 

Note: As a precaution this solution should always be prepared over a 
sink. 



47 





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